MOLECULAR MECHANISMS OF GLUCOCORTICOID HORMONE ACTION

了解基因表达的调控机制是至关重要的 发展和分化的理解，因此，癌症， 当这些过程出错时可能出现的其他疾病。 类固醇 激素调节的基因表达提供了一个极好的模型系统， 研究监管机制。小鼠乳腺肿瘤病毒 （MMTV）启动子已作为原型的研究开发的 糖皮质激素分子机制的概念框架 行动上虽然糖皮质激素调节元件（GRE）共识 序列已经提出，功能反应元件不能被 从序列数据可靠地预测。此外，更多的响应元素， 特别是那些介导负调节的基因， 不符合共识。我们的研究集中在一个系统的 突变的方法来定义糖皮质激素反应元件的糖皮质激素反应元件。 MMTV启动子。在上一个赠款期间，我们开发和 表征了GRE点突变体的文库。定量测定 60多个突变体的活动使我们能够改进我们的 GRE的概念。我们建议补充生物活性 受体与突变HRE结合的体外分析数据。的 结合研究的结果将与功能结合起来。 活性分析，以组装受体关键残基图谱 结合和激素反应。这些映射数据将产生洞察力， 受体-DNA相互作用的分子细节， 识别受体进行氢键接触的可能位点， 使其能够区分HRE序列。因为MMTV的发起人现在已经 被证明对至少4种不同类型的类固醇有反应， 通过将这些分析扩展到 其他调节该启动子的类固醇受体系统。等 比较分析将揭示不同的受体如何区分 识别序列的不同特征。研究重点是 典型的积极GRE将通过类似的分析来补充 增殖素基因的负GRE是一个受体识别位点， 与MMTV GRE仅有弱同源性。最后，提出了一些方法， 研究体内受体DNA相互作用，不仅与GRE突变体， 而且在与类固醇无反应性相关的情况下 例如与细胞相关类固醇拮抗剂或类固醇抗性 转型

Understanding the mechanisms of regulation of gene expression is essential to a comprehension of development and differentiation, and thus, cancer and the other disorders that may arise when these processes go awry. Steroid hormone regulated gene expression provides an excellent model system with which to investigate regulatory mechanisms. The mouse mammary tumor virus (MMTV) promoter has served as the prototype for studies developing the conceptual framework of the molecular mechanisms of glucocorticoid hormone action. Although glucocorticoid regulatory element (GRE) consensus sequences have been proposed, functional response elements cannot be reliably predicted from sequence data. In addition, more response elements, especially those mediating negative regulation, are being mapped which do not conform to the consensus. Our studies have focused on a systematic mutagenesis approach to defining the glucocorticoid response element of the MMTV promoter. During the previous grant period we developed and characterized a library of GRE point mutants. The quantitation of the activity of more than 60 of the mutants has allowed us to refine our conception of the GRE. We propose to complement the biological activity data with in vitro analyses of receptor binding to the mutant HREs. The results of the binding studies will be combined with the functional activity analyses to assemble a map of residues critical for receptor binding and for hormone response. These mapping data will yield insights as to molecular details of the receptor-DNA interaction particularly to identify the likely sites where receptors make hydrogen bond contacts that allow it to discriminate HRE sequences. Because the MMTV promoter has now been shown to respond to at least 4 different classes of steroids, the mutant library can be further exploited by extending these analyses to the other steroid-receptor systems that regulate this promoter. Such comparative analyses will reveal how the different receptors distinguish different features of the recognition sequence. The studies focusing on the prototypical positive GRE will be complemented by similar analyses on the negative GRE of the proliferin gene a receptor recognition site that bears only weak homology to the MMTV GRE. Finally, approaches are proposed to investigate receptor DNA interaction in vivo, not only with GRE mutants, but also under circumstances associated with steroid non-responsiveness e.g. steroid antagonists or steroid resistance associated with cell transformation.